Blood vessels grow in response to tissue injury and ischemia via angiogenesis, arteriogenesis and vasculogenesis. Transcriptional activation of a number of angiogenesis-related genes including VEGF, VEGF receptors such as flt-1 and neuropilin-1, and PDGF-B, and angiopoietin among others (Semenza (2000) Biochem. Pharmacol. 59:47-53) are regulated by hypoxia-inducible factor (HIF)-1α. Further, macrophages secrete numerous proteins including cytokines (IL-2 and TNF-α) and matrix metalloproteinases (Sunderkotter, et al. (1991) Pharmacol. Ther. 51:195-216; Gordon, et al. (1995) Curr. Opin. Immunol. 7:24-33; Arras, et al. (1998) Basic Res. Cardiol. 93:97-107). Among these is PR39, a cathelin-like “proline-and-arginine rich peptide” (PARP) originally isolated from pig intestine for its antimicrobial property (Agerberth, et al. (1996) Vet. Immunol. Immunopathol. 54:127-31; Agerberth, et al. (1991) Eur. J. Biochem. 202:849-54). PR39 and its homologs are found in the wound fluid of many animals as well as along the border of acute myocardial infarction (Gallo, et al. (1994) Proc. Natl. Acad. Sci. USA 91:11035-9). Secreted as a prepropeptide, the mature 39 amino acid C-terminal polypeptide chain (PR39) is produced by rapid cleavage of a canonical leader sequence (Gudmundsson, et al. (1995) Proc. Natl. Acad. Sci. USA 92:7085-9). PR39 crosses cell membranes readily, and is reported to bind to SH3 domains of cytosolic component of NADPH oxidase complex, protein p47phox15 and a signaling adaptor protein p130Cas9 (Gudmundsson, et al. (1995) supra; Chan & Gallo (1998) J. Biol. Chem. 273:28978-85; Chan, et al. (2001) J. Invest. Dermatol. 116:230-5).
PR39 is composed of 39 amino acids and induces angiogenesis and reduces inflammation in mouse models. PR39 is suggested to induce angiogenesis by acting along the VEGF and FGF pathway. PR39 also selectively inhibits the degradation of proteins including HIF-1α and IκBα, presumably by binding to the α7 subunit of the 20S proteasome. U.S. Patent Application Publication No. 20040009463 discloses a method for PR39 peptide-mediated selective inhibition of IκBα, and certain PR39-derived oligopeptides. A peptide composed of the first eleven amino acid residues of PR39, namely PR11, is also able to inhibit the 20S proteasome, albeit with a reduced Ki.
Studies have shown that PR39 stimulates angiogenesis in vitro and in vivo. Transgenic expression in cardiac myocytes results in increased vessel numbers and reduced coronary resistance (Li, et al. (2000) Nat. Med. 6:49-55). These effects appear to derive from the inhibition of HIF-1α degradation, which results in increased VEGF expression. PR39 also increases the expression of FGFR1 and syndecan-4, another FGF-2 signaling protein (Volk, et al. (1999) J. Biol. Chem. 274:24417-24; Li, et al. (1997) Circ. Res. 81:785-96), suggesting that PR39 may also induce angiogenesis via FGF pathway. PR39 appears to function by binding to the non-catalytic α7 subunit of 20S proteasome and inhibiting the degradation of another key intracellular protein, NF-κB inhibitor, IκBα (Gao, et al. (2000) J. Clin. Invest. 106:439-48).
Non-lysosomal degradation of cellular proteins occurs by the action of E1, E2 and E3 enzymes that result in the tetra-ubiquitinylation of target proteins and their proteolysis by the enzymatic activities residing within the central chamber of the 20S proteasomes. Ubiquitin and ubiquitin-like proteins are responsible for regulating numerous cellular pathways including the cell division cycle, transcription, protein sorting in the secretory pathway, membrane protein transport, endocytosis, nuclear transport, and signal transduction. The identification and analyses of inhibitors of proteasome are, therefore, of immense value to treat a variety of diseases, e.g., cancer, autoimmune diseases, muscle wasting, and inflammation. One example of a successful proteasome-based drug is the boronate, bortesomib (VELCADE™). This, and a variety of other agents currently in clinical testing act by blocking the enzymatic activities resident within the 20S proteasome resulting in the cessation of proteolysis of all substrate proteins and triggering apoptosis.
Cylindrical 20S proteasomes of eukaryotes, at approximately 700 kDalton mass, are composed of two heptameric inner rings of β-subunits and two heptameric outer rings of α-subunits. Thus, it is not surprising that a majority of proteasome inhibitors target the active sites that are exclusively associated with the β-subunits. The α-subunits are not known to possess proteolytic activity. It is remarkable, then, that the association of PARP with the α7 subunit of 20S proteasome should function to selectively inhibit proteolytic degradation of polypeptide chains such as IκBα. Atomic force microscopic investigations reveal that the 20S proteasome undergoes a gross structural change upon binding PR39. However, this does not sufficiently explain the mechanism of selective inhibition by PARP or the basis of PARP-proteasome interactions.